Urinary trypsin inhibitor (xe2x80x9cUTIxe2x80x9d) is a glycoprotein that inhibits the enzyme reactivity of trypsin and xcex1-chymotrypsin, hyaluronidase, and creatine phosphokinase. UTI can be present in minute quantities in the urine of healthy individuals.
Trypsin inhibitor activity has been suggested for use in a screening test for diagnosing bacterial infection. When bacterial infections occur, white blood cells are mobilized, and the elastase activity of the white blood cells is activated. During the acute phase response, interleukin-1 induces the production of inter-xcex1-trypsin inhibitor, which is decomposed by the elastase activity into low molecular weight trypsin inhibitors. These trypsin inhibitors appear to act on the inflamed sites, showing anti-inflammatory and anti-shock activities before being excreted in the urine. Piette et al. (European J. Med. 1, 273 (1992)) reports that urinary trypsin inhibitor activity can be a useful marker, particularly in patients with fever of unknown origin or elevated erythrocyte sedimentation rate.
Quantitative changes in UTI are useful as an index of infection or inflammation. Kuwajima et al. (Clin. Biochem. 23, 167 (1990)) reports that the assay of UTI may be used for the clinical diagnosis of acute phase response. UTI levels are elevated under other circumstances such as malignant tumors, kidney disease, myocardial infarction and post surgery.
Serum C-reactive protein, sialic acid and erythrocyte sedimentation rate have been used as markers of infection and inflammation. However, all of these markers are serum-based, which requires a blood sample. Using blood samples requires time for coagulation, centrifugation, and separation of the blood sample before analysis.
Measuring UTI concentration has been accomplished several ways, including enzyme inhibition, antibody stains, latex agglutination methods and radioimmunoassay methods. Enzyme inhibition has been used to measure UTI concentration, and calorimetric enzyme substrates have been used to measure the extent of the inhibition. The method has been recently adapted to automated measurement on clinical analyzers (S. Kuwajima, et al., loc,. cit.). Such analytical techniques typically involve contacting the urine sample with a trypsin substrate attached to a chromophore at either arginine or lysine, because trypsin cleaves arginine and lysine. The concentration of UTI in the urine sample is inversely proportional to the intensity of the colored response of the chromophore since UTI inhibit trypsin activity according to their concentration in the fluid test sample.
Several calorimetric and fluorogenic trypsin substrates are commercially available, including Nxcex1-benzoyl-L-arginine p-nitroanilide (BAPNA), Nxcex1-benzoyl-D,L-arginine xcex2-naphthylamide (BANA) and Nxcex1-benzoyl-L-arginine-7-amido-4-methylcournarin.
Known indicating trypsin substrates are aromatic amides of Nxcex1-protected arginine. When trypsin hydrolyzes these known substrates, the amide bond is cleaved and an aromatic amine is released. In the case of BAPNA, the amide bond is cleaved and yellow-colored p-nitroaniline is liberated and measured with a spectrophotometer. With BANA, 2-amino-naphthalene is produced, and it is detected by diazotization and coupling with N-(1-naphthyl)-ethylenediamine to form an azo dye (Goldberg, et al., Cancer 11, 283 (1958)). 7-Amino-4-methylcoumarin is released by hydrolysis of Nxcex1-benzoyl-L-arginine-7-amido-4-methylcoumarin, and this fluorescent product is measured with a fluorometer. These substrates are used for measuring trypsin activity in liquid-phase assays but are not well suited for use in dry-phase formats, such as dip-sticks, which are typically read visually or with simple reflectance instruments.
Aromatic esters of arginine are not known to those of skill in the art as trypsin substrates. Esters are much more labile toward hydrolysis than amides, and are often incorporated into protease substrates in place of amides to give more sensitive, easily hydrolysed analogs. They are also more prone to non-enzymatic hydrolysis by nucleophiles. This is significant for arginine esters, which have the nucleophilic guanidino group as part of their structure. Gray, et al. (Enzyme Microb. Technol. 5, 137 (1983)) states that efforts to prepare the Nxcex1-benzoyl-arginine esters of 2-hydroxynaphthol and 7-hydroxy-4-methylcoumarin were unsuccessful because of the lability of the ester group.
A trypsin substrate is needed that addresses the short-comings of prior art including, among other things, the requirement of a blood sample.
This invention provides aromatic esters of Nxcex1-(xcex1 amino group) and NG-(guanidino group) bis-protected arginine that are trypsin substrates. Surprisingly, trypsin hydrolyzes esters of arginine with protecting groups on the guanidino moiety. The esters of the present invention may be used to produce visible colors in dry-phase analytical elements to detect quantities of UTI in biological sample such as urine.
In one aspect of the invention, a compound of the formula (I) comprises: 
wherein R1 is a protecting group for Nxcex1, R2 is a protecting group for NG; and R3 is aryl; and wherein the compound of formula (I) is a trypsin substrate such that trypsin cleaves the Oxe2x80x94C single bond, which liberates R3xe2x80x94OH.
In another aspect of the invention, a diagnostic device comprises a carrier matrix and a compound of the formula (I).
In another aspect of the invention, a method of preparing a diagnostic device comprises (a) contacting a carrier matrix with a buffer solution, (b) drying the carrier matrix, and (c) contacting the carrier matrix with a solution comprising the trypsin substrate of formula (I).
In still another aspect of the invention, a method for detecting levels of urinary trypsin inhibitor in a biological sample comprises (a) contacting a biological sample with a predetermined amount of trypsin, a predetermined amount of a diazonium salt, and a diagnostic device comprising a trypsin substrate of the formula (I) wherein R1 is a protecting group for Nxcex1; R2 is a protecting group for NG; and R3 is aryl; and wherein the compound of formula (I) is a trypsin substrate such that trypsin cleaves the Oxe2x80x94C single bond, which liberates R3xe2x80x94OH; and wherein the compound R3xe2x80x94OH reacts with a diazonium salt to form a visible color such that the greater the intensity of the color, the less urinary trypsin inhibitor is in the biological sample.
In still another aspect of the invention, a diagnostic kit for determining the presence of urinary trypsin inhibitor in a biological fluid comprises trypsin and a trypsin substrate of the formula (I).
The present invention provides the foregoing and other features, and the advantages of the invention will become further apparent from the following detailed description of the presently preferred embodiments. The detailed description is merely illustrative of the invention and does not limit the scope of the invention, which is defined by the appended claims and equivalents thereof.
Definition of Terms
xe2x80x9cAlkylxe2x80x9d as used herein is the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups and cycloalkyl groups. Particularly preferred alkyl substituents include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl, pentyl, hexyl, cyclohexyl, etc. Unless the number of carbons is otherwise specified, xe2x80x9clower alkylxe2x80x9d as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. The aliphatic cyclic groups can be single or polycyclic containing between about 1 to 12 carbons per ring, but preferably between 1 and 9 carbons per ring.
xe2x80x9cArylxe2x80x9d as used herein includes 5-15 membered aromatic monocyclic or fused polycyclic moieties which may include from zero to four heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen. For example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthylene, benzothiazole, benzothiaphene, benzofuran, indole, quinoline, etc. The aryl group can be substituted at one or more positions with halo, alkyl, hydroxy, alkoxy, alkoxy carbonyl, haloalkyl, cyano, amino sulfonyl, aryl, sulfonyl, aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substituted or unsubstituted substituents, provided the substituent does not interfere with the ability of the composition of formula (I) to hydrolyze in the presence of trypsin.
xe2x80x9cHeteroarylxe2x80x9d as used herein is a mono-, bi- or tricyclic, xe2x80x94Nxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 heteroaryl substituent, such as benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole and thiophene.
xe2x80x9cProtecting groupxe2x80x9d as used herein is a group that is used to protect a functional group from unwanted reactions. After application, the protecting group can be removed.
The Trypsin Substrate
The trypsin substrates of the present invention include aromatic esters of Nxcex1,NG-bis-protected-arginine and Nxcex1,NG-bis-protected-arginine derivatives. When the esters are hydrolyzed by trypsin, an aromatic alcohol is liberated, producing a readily detectable signal if trypsin is present in the biological sample.
The arginine esters are described generically as compounds of formula (I): 
wherein R1is a protecting group for Nxcex1; R2 is a protecting group for NG; and R3 is aryl; and wherein the compound of formula (I) is a trypsin substrate such that trypsin cleaves the Oxe2x80x94C single bond; which liberates R3xe2x80x94OH. In one embodiment, R3xe2x80x94OH is optically distinguishable from the compound of formula (I). In this embodiment, R3xe2x80x94OH is preferably visually distinct (using only the naked eye) from the compound of formula (I). Alternatively, R3xe2x80x94OH can be optically distinguishable from the compound of formula (I) using analytical instrumentation.
In another embodiment, R3xe2x80x94OH reacts with a diazonium salt to form a visible color.
Esters of p-nitrophenol, which produce a yellow color upon hydrolysis, are useful for trypsin detection in samples with little or no intrinsic color. In colorful biological samples, such as urine or blood serum, however, interference from endogenous colored constituents is minimized by using a substrate that produces an intense absorption in the visible region of the spectrum, preferably  greater than 500 nm. For this reason, the preferred substrates are derivatives of aromatic alcohols that readily form intensely-colored azo dyes when coupled with aromatic diazonium salts.
These arginine esters are preferably prepared by esterification of the carboxyl moiety of an Nxcex1,NG-protected-arginine with an aromatic alcohol. Preferably, the ester and alcohol possess different optical properties or chemical reactivities. For example, esters of p-nitrophenol are colorless, but the free phenol is yellow at pH greater than 7. Esters of 7-hydroxy-4-methylcoumarin are non-fluorescent, while the free hydroxy-coumarin is highly fluorescent. Esters of 3-hydroxy-5-phenylpyrrole are unreactive toward aromatic diazonium salts like 2-methoxy-4-morpholinobenzenediazonium chloride (MMBD), whereas 3-hydroxy-5-phenylpyrrole quickly reacts with MMBD to produce a brightly-colored azo dye.
Any of these optical or chemical differences may be used to detect UIT in a biological sample.
Protecting Groups for Nxcex1
R1 is a protecting group for Nxcex1. Preferred Nxcex1 protecting groups are stable and render the Nxcex1 function inert under the conditions employed in the reactions involved in making the trypsin substrate and in the reactions involved where trypsin cleaves the Oxe2x80x94C single bond of the ester functional group. The species of the Nxcex1 protecting group used is not critical so long as the derivatized amino group is stable to the conditions of the subsequent reactions and does not interfere with the ability of the composition to hydrolyze in the presence of trypsin.
Suitable protecting groups for Nxcex1 include, but are not limited to, carbamates, amides and aryl sulfonamides. Carbamates include the xcfx84-butoxycarbonyl (xcfx84-BOC) group, the carbobenzyloxy (CBZ) group and others known in the art. Amide protecting groups include lower alkyl amides such as the acetyl group and aryl amides such as the benzoyl group. Suitable aryl sulfonamide groups include the benzene sulfonyl group, the xcfx81-toluenesulfonyl (tosyl) group and others known in the art. These and other suitable protecting groups may include those listed in the chapter entitled xe2x80x9cProtection for the Amino Groupxe2x80x9d of the third edition (April 1999) of xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d by Green and Wuts, which is hereby incorporated by reference.
Protecting Groups for NG 
R2 is a protecting group for NG, also known as the guanidine N of arginine. The presence of a protecting group on the NG reduces the nucleophilicity of the guanidine moiety. The protecting group protects the ester from non-enzymatic hydrolysis. Further, the NG-protecting group does not completely inhibit enzymatic hydrolysis, so that these arginine esters are stable and useful as trypsin substrates.
Preferred NG protecting groups are stable and render the NG function inert under the conditions employed in the reactions involved in making the trypsin substrate and in the reactions involved where trypsin cleaves the Oxe2x80x94C single bond of the ester functional group.
Suitable protecting groups for NG include, but are not limited to, nitro, arene sulfonyl compounds, and carbonyl derivatives. A non-limiting list of suitable NG protecting groups may include nitro, tosyl, p-methoxybenzenesulfonyl, carbonbenzyloxy, benzoyl, and similarly-structured protecting groups.
The Aryl Moiety that Forms the Aromatic Alcohol
R3 is aryl as defined above. When trypsin hydrolyzes the substrate, trypsin cleaves the Oxe2x80x94C single bond in the ester moiety of the compound of formula (I). This causes the formation of the compound R3xe2x80x94OH. R3 must be stable so that it does not form the compound R3xe2x80x94OH absent the compound for formula (I) being cleaved by trypsin.
In one embodiment, R3xe2x80x94OH is optically distinguishable from the compound of formula (I). In another embodiment, R3xe2x80x94OH reacts with a diazonium salt to form a visible color, preferably, a color that is different from the color of the biological sample.
Generally R3 can be any aryl compound such that the compound R3xe2x80x94OH, when formed by trypsin cleaving the compound of formula (I), can be optically distinguished from the compound of formula (I) or reacts with a diazonium salt to form a color in the visible region. Preferably, R3 comprises a heterocyclic aromatic moiety. Preferably, the heterocycle is in a fused ring system and the heteroatom is selected from the group consisting of N or O. Preferred R3 groups may include, but are not limited to, phenylpyrrole and derivatives thereof, coumarin and derivatives thereof, phenylthlophene and derivatives thereof, indole and derivatives thereof, and 2-phenyl-5H-thiazol and derivatives thereof.
Diazonium Salts
A diazonium salt is generally an organic salt of a compound having a diazonium radical, a illustrated by the general structure:
R4xe2x80x94N2+Anxe2x88x92
Wherein R4 is an aryl moiety as defined previously and Anxe2x88x92 is an anion. An represents any suitable anion such as halide (for example chloride, bromide, fluoride and iodide), tetrafluoroborate, chlorozincate, hemizinc chloride, nitrate, perchlorate, xcfx81-toluenesulfonate and others readily apparent to one skilled in the art.
Other contemplated diazonium salts incorporate the anion in R4 and are zwitterions having the structure: 
wherein Dxe2x88x92 is an anion. Preferred anions include SO3xe2x88x92, CO2xe2x88x92, and PO3=. G is independently H, C1-6 alkyl, or in which the two G moieties together form a fused ring system. B is H or OH.
Any diazonium salt that reacts with the aromatic alcohol (R3xe2x80x94OH) to form a color in the visible region may be used with the trypsin substrate. Preferred diazonium salts are those that do not readily react with other urinary components during the detection of UTI. A non-limiting list includes 2,4-dimethoxybenzene-diazonium tetrafluoroborate, 4-methoxynaphthalene-1-diazonium tetrafluoroborate, 2,5-dimethoxy-4-dimethylaminobenzene-diazonium tetrafluoroborate, 4-dimethylaminobenzenediazonium tetafluoroborate, 2-methoxy-4-(N-pyrrolidino)-benzenediazonium tetrafluoroborate, 2-methoxy-4-(N-piperidino)-benzene-diazonium tetrafluoroborate, 2,6-dimethoxy-4-(N-morpholino)-benzenediazonium tetrafluoroborate, 4-methoxy-2-(N-morpholino)-benzenediazonium hemizinc chloride (MMBD), 2-methoxy-4-[N-(Nxe2x80x2-methyl)piperazino]-benzenediazonium tetrafluoroborate, 2-methoxy-4-(N-thiomorpholino)-benzendiazonium tetrafluoroborate and the like.
Preferred zwitterionic diazonium salts include 1-diazonphthalene-4-sulfonate, 1-diazo-2-naphthol-4-sulfonate, 1-diazo-2-naphthol-4,6-disulfonate, 1-diazophenyl-3-carbonate as disclosed in U.S. Pat. No. 4,637,979 (Skjold, et al.) which is hereby incorporated by reference.
Many diazonium salts useful herein are available from a number of commercial sources, and those not readily available can be prepared by a skilled organic chemist using available reagents and well-known procedures.